Applied Methods to Assess the Antimicrobial Activity of Metallic-Based Nanoparticles

Abstract

With the rise of antibiotic resistance, the drive to discover novel antimicrobial substances and standard testing methods with the aim of controlling transmissive diseases are substantially high. In healthcare sectors and industries, although methods for testing antibiotics and other aqueous-based reagents are well established, methods for testing nanomaterials, non-polar and other particle-based suspensions are still debatable. Hence, utilities of ISO standard validations of such substances have been recalled where corrective actions had to be taken. This paper reports a serial analysis obtained from testing the antimicrobial activities of 10 metallic-based nanomaterials against 10 different pathogens using five different in vitro assays, where the technique, limitation and robustness of each method were evaluated. To confirm antimicrobial activities of metallic-based nanomaterial suspensions, it was found that at least two methods must be used, one being the agar well diffusion method, which was found to be the most reliable method. The agar well diffusion method provided not only information on antimicrobial efficacy through the size of the inhibitory zones, but it also identified antimicrobial ions and synergistic effects released by the test materials. To ascertain the effective inhibitory concentration of nanoparticles, the resazurin broth dilution method is recommended, as MIC can be determined visually without utilising any equipment. This method also overcomes the limit of detection (LoD) and absorbance interference issues, which are often found in the overexpression of cell debris and nanoparticles or quantum dots with optical profiles. In this study, bimetallic AgCu was found to be the most effective antimicrobial nanoparticle tested against across the bacterial (MIC 7 µg/mL) and fungal (MIC 62.5 µg/mL) species.

Keywords: antimicrobial nanoparticles; copper; live–dead assay; minimum inhibitory concentration (MIC); resazurin; silver.

Figure 1

(a–c): 24 h kinetic growth curve of microbes after exposing (a) C. albicans, (b) E. coli, and (c) S. aureus with AgCu nanoparticles at serial concentrations between 62.5 µg/mL and 0.5 µg/mL. Each data point represents the mean value of quadruplicate time measurements at OD₆₀₀. The corresponding standard deviations can be found in supplementary data Figure S4a–c.

Figure 2

Percentage cell viability of microbes after exposures of different concentrations of AgCu nanoparticles (100 µg/mL and the corresponding MIC) over period of five hours. Results represent three areas of slide count of two replicates.

Figure 3

Fluorescent observation of C. albicans cell viability after AgCu nanoparticles exposure over five hours at MIC 62.5 µg/mL and concentration of 100 µg/mL. Green fluorescence represents live viable cells and red fluorescence represents dead cells. Intermediate colours yellow and orange indicated partially damaged cells.

Figure 4

Fluorescent observation of E. coli cell viability after exposure of AgCu nanoparticles over five hours at MIC 7 µg/mL and concentration of 100 µg/mL. Green fluorescence represents live viable cells and red fluorescence represents dead cells.

Figure 5

Fluorescent observation of S. aureus cell viability after exposure of AgCu nanoparticles over five hours at MIC 31 µg/mL and concentration of 100 µg/mL. Green fluorescence represents live viable cells and red fluorescence represents dead cells.